EXPRESSION AND BIOCHEMICAL-CHARACTERIZATION OF IRON REGULATORY PROTEIN-1 AND PROTEIN-2 IN SACCHAROMYCES-CEREVISIAE

Citation
Jd. Phillips et al., EXPRESSION AND BIOCHEMICAL-CHARACTERIZATION OF IRON REGULATORY PROTEIN-1 AND PROTEIN-2 IN SACCHAROMYCES-CEREVISIAE, Biochemistry, 35(49), 1996, pp. 15704-15714
Citations number
64
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
35
Issue
49
Year of publication
1996
Pages
15704 - 15714
Database
ISI
SICI code
0006-2960(1996)35:49<15704:EABOIR>2.0.ZU;2-K
Abstract
Iron-regulatory proteins (IRPs) 1 and 2 are cytosolic RNA-binding prot eins that bind to specific stem-loop structures, termed iron-responsiv e elements (TREs) that are located in the untranslated regions of spec ific mRNAs encoding proteins involved in iron metabolism. The binding of IRPs to IREs regulates either translation or stabilization of mRNA. Although IRP1 and IRP2 are similar proteins in that they are ubiquito usly expressed and are negatively regulated by iron, they are regulate d by iron by different mechanisms. IRP1, the well-characterized IRP in cells, is a dual-function protein exhibiting either aconitase activit y when cellular iron is abundant or RNA-binding activity when cellular iron is scarce. In contrast, IRP2 lacks detectable aconitase activity and functions exclusively as an RNA-binding protein. To study and com pare the biochemical characteristics of IRP1 and IRP2, we expressed wi ld-type and mutant rat IRP1 and IRP2 in the yeast Saccharomyces cerevi sine. IRP1 and IRP2 expressed in yeast bind the IRE RNA with high affi nity, resulting in the inhibition of translation of an IRE-reporter mR NA. Mutant IRP2s lacking a 73 amino acid domain unique to IRP2 and a m utant IRP1 containing an insertion of this domain bound RNA, but lacke d detectable aconitase activity, suggesting that the presence of this domain prevents aconitase activity. Like LRP1, the RNA-binding activit y of IRP2 was sensitive to inactivation by N-ethylmaleimide (NEM) or 5 ,5'-dithiobis(2-nitrobenzoic acid) (DTNB), indicating IRP2 contains a cysteine(s) that is (are) necessary for RNA binding. However, unlike I RP1, where reconstitution of the 4Fe-4S cluster resulted in a loss in RNA-binding activity, the RNA-binding activity of IRP2 was unaffected using the same iron treatment. These data suggested that IRP2 does not contain a 4Fe-4S cluster similar to the cluster in IRP1, indicating t hat they sense iron by different mechanisms.